Embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocysts, are capable of self-renewal and differentiation along different cell lineages, a property known as pluripotence (1
). Because ESC differentiation can be directed into particular lineages with specialized functional properties for tissue repair and replacement, they are considered to be an excellent resource for regenerative medicine (2
). Three major obstacles associated with using ESCs for regenerative medicine are: (1
) precise and controlled differentiation of ESCs toward a well-defined lineage; (2
) isolation of homogeneous populations of stably differentiated cells that are fully functional; and (3
) retaining the expansion potential.
A common approach is to pre-differentiate ESCs into three-dimensional cell aggregates known as embryoid bodies (EBs). These bodies contain cells from all three germ layers, mesoderm, ectoderm, and endoderm, and can self-renew and differentiate into different cell types. Embryoid bodies can be produced in three main ways: (1
) hanging drop; (2
) methylcellulose hydrogel; and (3
) suspension culture. The most convenient and efficient of these is suspension culture of ESCs in non-adherent plates (3
). This approach produces large amounts of EBs in a short time, which is advantageous for high-throughput drug screening and tissue engineering. However, an important consideration is embryoid body size, which may affect the outcome and properties of differentiated cells. For example, uncontrolled overgrowth of EBs may result in cavity formation due to apoptosis, after which EBs eventually become cystic and contain fluid.
Methods for developing differentiated endothelial cells from EBs are well established and the corresponding gene expression patterns are well characterized (4
). Embryonic stem cells have been directly differentiated toward specific lineages without first making EBs by using conditioned medium for endothelial differentiation of ESC in collagen IV plates (7
Large-scale changes in gene expression accompany the initial differentiation of ESCs into EBs, and subsequent large-scale changes in gene expression are linked to lineage-specific differentiation of EBs along mesenchymal, epithelial, neural, or hematopoietic lineages (9
). Embryoid bodies can be directed to give rise to hemangioblasts, which subsequently undergo further differentiation into either hematopoietic or endothelial cells. Hemangioblasts have been widely used to study the expression of transcription factors that control EC lineage and recapitulate many aspects of vascular development in vivo (12
). The expression of vascular endothelial growth factor receptor-2 (VEGF-R2), also known as Flk-1, in mice, is a mesodermal indicator and the earliest functional marker for hemangioblasts (13
After EBs are formed and exposed to endothelial differentiation medium containing VEGF, a heterogeneous cell population, including mesenchymal, hematopoietic, and epithelial cells, emerges. Only a subset of these cells will differentiate toward endothelial cells (less than 2 % with our current method). Therefore, once differentiation has been induced the endothelial cell population must be isolated and purified for further expansion and analysis.
Herein, we describe our methods for endothelial differentiation of mouse ESCs (mESCs) and FACS-based isolation and expansion of the resulting cell populations that express the endothelial-specific markers Flk-1 and VE-Cadherin on their surface.